The invention concerns polymerase chimeras which are composed of amino acid fragments representing domains and which combine properties of naturally occurring polymerases that are advantageous with regard to a particular application. It has surprisingly turned out that the domains from the various enzymes are active in the chimeras and exhibit a cooperative behaviour. The present invention especially concerns those polymerase chimeras in which the domains having polymerase activity and domains having 3xe2x80x2-5xe2x80x2exonuclease activity are derived from different enzymes. Such chimeras can also have RT activity. In addition the present invention concerns a process for the production of the chimeras according to the invention and the use of these chimeras for the synthesis of nucleic acids e.g. during a polymerase chain reaction. Moreover the present invention concerns a kit which contains the polymerase chimeras according to the invention.
According to Braithwaite, D. K. and Ito, J. (1993) Nucl. Acids Res. 21, 787-802 DNA polymerases are divided according to the correspondence in their amino acid sequences into three main families with subclasses. Joyce, C. M. and Steitz, T. A. (1994) Annu. Rev. Biochem. 63, 777-822 give a summary of the motifs and conserved amino acids that were found. In prokaryotes the main distinction is made between three polymerases: polymerase I, II and III. These polymerases differ with regard to their function in the cell and with regard to their properties. DNA polymerase I is considered to be a repair enzyme and frequently has 5xe2x80x2-3xe2x80x2 as well as 3xe2x80x2-5xe2x80x2 exonuclease activity. Polymerase II appears to facilitate DNA synthesis which starts from a damaged template strand and thus preserves mutations. Polymerase III is the replication enzyme of the cell, it synthesizes nucleotides at a high rate (ca. 30,000 per minute) and is considered to be very processive. Polymerase III has no 5xe2x80x2-3xe2x80x2 exonuclease activity. Other properties of polymerases are due to their origin such as e.g. thermostability or processivity.
Particular properties of polymerases are desirable depending on the application. For example thermostable, high-fidelity (i.e. polymerases with proof-reading activity), processive and rapidly synthesizing polymerases are preferred for PCR. Enzymes are preferred for sequencing which do not discriminate much between dideoxy and deoxy nucleotides. In contrast the proof-reading activity of polymerases, i.e. 3xe2x80x2-5xe2x80x2 exonuclease activity, is not desirable for sequencing. For some applications e.g. PCR it is desirable that the polymerase has no or little 5xe2x80x2-3xe2x80x2 exonuclease activity (5xe2x80x2 nuclease activity).
Polymerases can also differ in their ability to accept RNA as a template i.e. with regard to their reverse transcriptase (RT) activity. The RT activity may be dependent on the presence of manganese or/and magnesium ions. It is often desirable that the RT activity of the polymerase is independent of manganese ions since the reading accuracy of polymerase is decreased in the presence of manganese ions. Polymerases additionally differ in their processivity which is also a desirable property for many applications.
There is therefore a need to optimize the properties of polymerases with regard to a particular application. In the past this was often achieved by introducing mutations or by deleting functions of the polymerases.
Thus for example the 5xe2x80x2-3xe2x80x2 exonuclease activity was abolished by introducing mutations (Merkens, L. S. (1995) Biochem. Biophys. Acta 1264, 243-248) as well as by truncation (Jacobsen, H. (1974) Eur. J. Biochem. 45, 623-627; Barnes, W. M. (1992) Gene 112, 29-35). The ability of polymerases to discriminate between dideoxy and deoxynucleotides was reduced by introducing point mutations (Tabor S. and Richardson, C. C. (1995) Proc. Natl. Acad. Sci. 92, 6339-6343). Tabor and Richardson describe the construction of active site hybrids.
The object to provide polymerases with optimized properties was achieved by the present invention for the first time by producing polymerase chimeras by exchanging domains that are structurally and functionally independent of one another. Domains in the sense of the present invention are understood as regions which contain all essential centres or all functionally important amino acids such that the domains essentially retain their function. It is therefore also possible to exchange only parts i.e. functioning fragments of domains. Thus these domains can be referred to as functional amino acid fragments in the sense of the present invention. Furthermore the chimeras can be additionally modified by mutations or truncations. If it appears to be advantageous it is also possible to introduce mutations into the chimeras which further optimize their properties with regard to the respective application. Thus for example mutations can be introduced which reduce the ability of the polymerases to discriminate between dideoxy and deoxy nucleotides. Alternatively desired properties such as processivity can be strengthened or introduced by introducing mutations or by truncation. The introduction of mutations or truncations can also abolish undesired properties e.g. the 5xe2x80x2 nuclease activity.
Thus polymerase chimeras are a subject matter of the present invention which combine advantageous properties of naturally occurring polymerases with regard to a particular application. The polymerase chimeras according to the invention are composed of functional amino acid fragments of different enzymes which preferably represent domains of different enzymes. The invention surprisingly showed that the domains from the different enzymes are active in the chimera and exhibit a cooperative behaviour between the domains. The present invention also concerns general processes for the production of polymerase chimeras with optimized properties. This process according to the invention thus enables a chimera to be designed from an arbitrary combination of enzymes by exchanging domains. It is additionally preferred that the interactions at the sites of contact between the domains are further harmonized by various methods. This can for example lead to an increase in the thermostability of the chimeras. A further subject matter of the invention is a kit for the synthesis of nucleic acids which contains a chimera according to the invention.
Thermostable DNA polymerases with proof-reading function are being increasingly used in practice for PCR. The use of mixtures of Taq polymerase and thermostable proof-reading DNA polymerase (such as Pfu, Pwo, Vent polymerase) has proven to be particularly successful for the amplification of long DNA molecules. Thus a further subject matter of the present invention was to combine the high processivity and thermostability of Taq polymerase with the 3xe2x80x2-5xe2x80x2 exonuclease activity of another DNA polymerase in one enzyme. Hence the present invention especially concerns thermostable polymerase chimeras which have a processivity which corresponds to at least that of Taq polymerase and have a low error rate when incorporating nucleotides into the polymer chain during amplification due to the presence of a 3xe2x80x2-5xe2x80x2 exonuclease activity (proof-reading activity). The combination of these two properties enables for example a chimera to be generated which is able to make long PCR products i.e. nucleic acid fragments which are larger than 2 kb. The chimera according to the invention is also suitable for amplifying shorter fragments.
The present invention therefore concerns in particular a polymerase chimera which is composed of functional amino acid fragments of two different polymerases wherein the first or the second polymerase has 3xe2x80x2-5xe2x80x2 exonuclease activity and the polymerase chimera has 5xe2x80x2-3xe2x80x2 polymerase activity as well as 3xe2x80x2-5xe2x80x2 exonuclease activity. The polymerases can be naturally occurring or recombinant polymerases. The polymerase chimera according to the invention can be composed of functional amino acid fragments from two or several different polymerases. The polymerase chimera according to the invention can be composed of two or several functional amino acid fragments from the different polymerases. The amino acid sequence of the fragment can correspond to the naturally occurring sequence of the polymerase or to a sequence modified by mutations.
The amino acid fragments from which the polymerase chimera is constructed preferably each correspond to functional polymerase domains of the first or second polymerase. A functional polymerase domain in the sense of the present invention is a region which contains all amino acids that are essential for the activity and is abbreviated as domain in the following.
The present invention concerns in particular a polymerase chimera composed of functional amino acid fragments (in short domains) from at least two different polymerases wherein the domain having polymerase activity is homologous to one polymerase and the domain having 3xe2x80x2 exonuclease activity is homologous to another polymerase. Moreover, this chimera can additionally have 5xe2x80x2 exonuclease activity in which case the domain having 5xe2x80x2 exonuclease activity can be homologous to the first or to the second polymerase. However, it is also possible that the 5xe2x80x2 exonuclease domain is partially or completely deleted or has point mutations. The polymerase chimera according to the invention can additionally have reverse transcriptase (RT) activity.
It is additionally preferred that a part of the amino acid fragments of the polymerase chimeras corresponds to a part of the amino acid sequence of Taq polymerase.
The polymerase whose domain or amino acid fragment having 3xe2x80x2-5xe2x80x2 exonuclease activity has been incorporated into the chimera can for example be a Pol-I type polymerase or also a Pol-II type polymerase. Representatives of the Pol-I type polymerase with 3xe2x80x2-5xe2x80x2 exonuclease activity are for example Escherichia coli polymerase (Ec.1), Salmonella polymerase I, Bacillus polymerase I, Thermosiphon polymerase I and Thermatoga neapolitana polymerase (Tne). Representatives of the Pol-II type polymerase with 3xe2x80x2-5xe2x80x2 exonuclease activity are for example Pyrrococcus woesie polymerase (Pwo), Pyrococcus furiosus polymerase (Pfu), Thermococcus litoralis polymerase (Tli), Pyrodictum abyssi. 
Representatives of Pol-I type and Pol-II type polymerases which were mentioned as examples are described in more detail in the following:
The Taq DNA polymerase from Thermus aquaticus (Taq polymerase), Escherichia coli DNA polymerase I (E. coli polI) and Thermotoga neapolitana DNA polymerase (Tne polymerase) are bacterial DNA polymerases from the A family. They are DNA polymerases of the polI type since the various enzymatic activities are located in the various domains in a relatively similar manner to that found in E. coli polI. The Pyrococcus woesi DNA polymerase (Pwo polymerase) is, like Thermococcus litorales DNA polymerase (Vent(trademark) polymerase) and Pyrococcus furiosus DNA polymerase (Pfu polymerase), an archaebacterial DNA polymerase of the B family.
Taq polymerase is described by Chien, A. et al. (1976) J. Bacteriol. 127, 1550-1557, Kaledin, A. S. et al. (1980) Biokhimiya 45, 644-651 and Lawyer, F. C. et al. (1989) J. Biol. Chem. 264, 6427-6437. It was originally isolated from the thermophilic eubacterium Thermus aquaticus and later cloned in E. coli. The enzyme has a molecular weight of 94 kDa and is active as a monomer. Taq polymerase is suitable for use in the polymerase chain reaction (PCR) since it has a high thermal stability (half life of 40 minutes at 95xc2x0 C./5 minutes at 100xc2x0 C.) and a highly processive 5xe2x80x2-3xe2x80x2 DNA polymerase (polymerisation rate: 75 nucleotides per second). Apart from the polymerase activity, a 5xe2x80x2 nuclease activity was detected by Longley et al. (1990) Nucl. Acids Res. 18, 7317-7322. The enzyme has no 3xe2x80x2-5xe2x80x2 exonuclease activity so that errors occur during the incorporation of the four deoxyribonucleotide triphosphates to successively extend polynucleotide chains which interfere with the gene amplification (error rate: 2xc3x9710xe2x88x924 errors/base, Cha, R. S. and Thilly, W. G. (1993) PCR Methods Applic. 3, 18-29). The tertiary structure of Taq polymerase has been known since 1995 (Kim et al., 1995, Korolev et al., 1995).
E. coli polI is described in Kornberg, A. and Baker, T. A. (1992) DNA Replication, 2nd edition, Freeman, New York, 113-165. The enzyme has a molecular weight of 103 kDa and is active as a monomer. E. coli polI has 5xe2x80x2 nuclease activity and 5xe2x80x2-3xe2x80x2 DNA polymerase activity. In contrast to Taq polymerase, it additionally has a 3xe2x80x2-5xe2x80x2 exonuclease activity as a proof-reading function. E. coli polI and its Klenow fragment (Jacobsen, H. et al. (1974) Eur. J. Biochem. 45, 623-627) were used for PCR before the introduction of Taq polymerase. However, due to their low thermal stability they are less suitable since they have to be newly added to each cycle. The tertiary structure of the Klenow fragment of E. coli polI has been known since 1983 (Brick, P. et al., (1983) J. Mol. Biol. 166, 453-456, Ollis, D. L. et al. (1985) Nature 313, 762-766 and Beese, L. S. et al. (1993) Science 260, 352-355).
Tne polymerase was isolated from the thermophilic eubacterium Thermotoga neapolitana and later cloned in E. coli. The amino acid sequence of the Tne polymerase is similar to that of Thermotoga maritima DNA polymerase (UITma(trademark) polymerase) (personal information from Dr. B. Frey). It has a high thermal stability, 5xe2x80x2 nuclease activity, 3xe2x80x2-5xe2x80x2 exonuclease activity and 5xe2x80x2-3xe2x80x2 DNA polymerase activity. A disadvantage is the low polymerisation rate compared with that of Taq polymerase. The UITma(trademark) polymerase which has a similar amino acid sequence is used for PCR if a high accuracy is required. Of the structure of Tne polymerase, only the amino acid sequence is known up to now (Boehringer Mannheim). However, the enzyme is homologous to E. coli polI so that, although the tertiary structure is unknown, homology modelling is possible.
Pfu polymerase was isolated from the hyper-thermophilic, marine archaebacterium Pyrococcus furiosus. It has a high thermal stability (95% activity after one hour at 95xc2x0 C.), 3xe2x80x2-5xe2x80x2 exonuclease activity and 5xe2x80x2-3xe2x80x2 DNA polymerase activity (Lundberg, K. S. et al. (1991) Gene 108, 1-6). The accuracy of the DNA synthesis is ca. 10 times higher than that of Taq polymerase. It is used for PCR if a high accuracy is required. Of the structure only the amino acid sequence is known up to now.
Pwo polymerase (PCR Applications Manual (1995), Boehringer Mannheim GmbH, Biochemica, 28-32) was originally isolated from the hyperthermophilic archaebacterium Pyrococcus woesi and later cloned in E. coli. The enzyme has a molecular weight of about 90 kDa and is active as a monomer. Pwo polymerase has a higher thermal stability than Taq polymerase (half life  greater than 2 hours at 100xc2x0 C.), a highly processive 5xe2x80x2-3xe2x80x2 DNA polymerase activity and a high 3xe2x80x2-5xe2x80x2 exonuclease activity which increases the accuracy of the DNA synthesis. The enzyme has no 5xe2x80x2 nuclease activity. The polymerisation rate (30 nucleotides per second) is less than that of Taq polymerase. The enzyme is used for PCR if a high accuracy is required. The accuracy of the DNA synthesis is more than 10 times higher than when using Taq polymerase.
Ath polymerase was isolated from the thermophilic archaebacterium Anaerocellum thermophilum and later cloned in E. coli. Ath polymerase has a high thermal stability and still has at least 90% of the original activity after an incubation of 30 min at 80xc2x0 C. in the absence of stabilizing detergents. The polymerase also has RT activity in the presence of magnesium ions. Ath polymerase is deposited at the xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbHxe2x80x9d, Mascheroder Weg 1b, D38124 Braunschweig DSM Accession No. 8995. The Ath polymerase has 5xe2x80x2-3xe2x80x2 polymerase activity, 5xe2x80x2-3xe2x80x2 exonuclease activity but no 3xe2x80x2-5xe2x80x2 exonuclease activity. Histidine tags or other purification aids can be additionally incorporated into the amino acid sequence of the polymerase chimeras to improve the purification.
There are four main methods for introducing a 3xe2x80x2-5xe2x80x2 exonuclease activity of a polymerase into another polymerase for example into Taq polymerase which are also a subject matter of the present invention:
1. Insertion of the 3xe2x80x2-5xe2x80x2 exonuclease region of another DNA polymerase by exchange of a molecular region of Taq polymerase
This approach is particularly suitable since the Taq polymerase is homologous to E. coli polI which is composed of domains which are functionally and structurally independent (Joyce, C. M. and Steitz, T. A. (1987) TIBS 12, 288-292) and can serve as a model for other DNA polymerases (Joyce, C. M. (1991) Curr. Opin. Struct. Biol. 1, 123-129). Suitable DNA polymerases for the exchange are those for which a 3xe2x80x2-5xe2x80x2 exonuclease activity has been demonstrated, whose DNA sequence is known and the gene coding for the 3xe2x80x2-5xe2x80x2 exonuclease activity is available. For a rational protein design based on model structures it is additionally advantageous that the 3xe2x80x2-5xe2x80x2 exonuclease region and the polymerase region are homologous to E. coli polI. The 3xe2x80x2-5xe2x80x2 exonuclease region preferably fits well into the structure of E. coli polI and adjoins the polymerase region of Taq polymerase. Further advantages are an elucidated tertiary structure with available structural data and high thermal stability of the protein.
The following DNA polymerases are thus for example suitable:
a. E. coli polI
Apart from thermal stability, E. coli polI fulfils all the above-mentioned conditions. The tertiary structure of the Klenow fragment is available in the Brookhaven data bank and, like Taq polymerase, it belongs to the A family of DNA polymerases. The identity in the amino acid sequence is 32%. Taking the known domain structure into consideration, the largest agreements are found in the N-terminal and in the C-terminal region of the two proteins (32% identity in the 5xe2x80x2 nuclease domains, 49% identity in the polymerase domains). The shorter Taq polymerase has several deletions in the region of the 3xe2x80x2-5xe2x80x2 exonuclease domain (14% identity in the 3xe2x80x2-5xe2x80x2 exonuclease domain and intermediate domain). Since E. coli polI is thermolabile and the interactions at the interface between the two domains in the chimeric protein are no longer optimal, it is probable that the protein chimera will also have a lower thermal stability than that of Taq polymerase. This can be redressed by subsequent modification of amino acids at the interface.
b. Thermostable DNA polymerases
Among the thermostable DNA polymerases with 3xe2x80x2-5xe2x80x2 exonuclease that are nowadays used for PCR, the Pwo polymerase, Pfu polymerase, Vent(trademark) polymerase, Tne polymerase and UITma(trademark) polymerase appear to be suitable for combination with the Taq DNA polymerase. The genes of the Pwo polymerase and the Tne polymerase are accessible (via the Boehringer Mannheim Company). The Pfu polymerase can be obtained from Stratagene Inc. The Tne polymerase is well suited for a rational protein design due to its homology to Taq polymerase and E. coli polI. When using the Pfu polymerase designs are only possible based on amino acid sequence alignments taking into consideration the known conserved amino acids and motifs that are essential for the function.
2. Modification of the Taq DNA polymerase in the intermediate domain
In order to insert a 3xe2x80x2-5xe2x80x2 exonuclease activity it is necessary to insert all amino acids that are essential for the activity into the structure. According to the present state of knowledge this applies in particular to the three motifs Exo I, Exo II and Exo III. The essential motifs must additionally be linked in a suitable manner in order to be placed in the spatial position necessary for catalysis.
It is also possible to modify the Taq DNA polymerase in the polymerase region. A de novo design of polymerases is also in principle conceivable.
The chimeras according to the invention can be additionally optimized by:
1. Removing the 5xe2x80x2 nuclease domain (possible also proteolytically) or subsequently inactivating the 5xe2x80x2 nuclease activity (described in Merkens, L. S. (1995) Biochem. Biophys. Acta 1264, 243-248)
2. Modification by point mutations or fragment exchange
3. Optimization of the structures at the interface of the chimeras
4. Optimization by random mutagenesis and/or random recombination with other polymerase genes (molecular evolution).
Examples of polymerase chimeras according to the invention are the following:
Taq DNA polymerase (M1-V307)E.coli DNA polymerase (D355-D501) Taq DNA polymerase (A406-E832)
Taq DNA polymerase (M1-P291)E.coli DNA polymerase (Y327-K511) Taq DNA polymerase (L416-E832)
Taq DNA polymerase (M1-P291)E.coli DNA polymerase (Y327-H519) Taq DNA polymerase (E424-E832): point mutation A643G; Ile455Val SEQ ID NO.:1
Taq DNA polymerase (M1-P291)E.coli DNA polymerase (Y327-V536) Taq DNA polymerase (L441-E832)
Taq DNA polymerase (M1-P291)E.coli DNA polymerase (Y327-G544) Taq DNA polymerase (V449-E832); SEQ ID NO.:2
Taq DNA polymerase (M1-P302)E.coli DNA polymerase (K348-S365) Taq DNA polymerase (A319-E347) E.coli DNA poly(N450-T505) Taq DNA polymerase (E410-E4832);
Taq DNA polymerase (M1-V307)Tne DNA polymerase (D323-D468) Taq DNA polymerase (A406-E832)
Taq DNA polymerase (M1-P291)Tne DNA polymerase (P295-I478) Taq DNA polymerase (L416-E832)
Taq DNA polymerase (M1-P291)Tne DNA polymerase (P295-E485) Taq DNA polymerase (E424-E832); silent mutation A1449C SEQ ID NO.:3
Taq DNA polymerase (M1-P291)Tne DNA polymerase (P295-V502) Taq DNA polymerase (L441-E832)
Taq DNA polymerase (M1-P291)Tne DNA polymerase (P295-G510) Taq DNA polymerase (V449-E832); silent mutation C1767T SEQ ID NO.:4
Taq DNA polymerase (M1-P302)Tne DNA polymerase (E316-D333) Taq DNA polymerase (A319-E347) Tne DNA polymerase (I381-M394) Taq DNA polymerase (R362-L380) Tne DNA polymerase (E415-T472)Taq DNA polymerase (E410-E832);
G308D/V310E/L352N/L356D/E401Y/R305D
Taq DNA polymerase (1-291)Pfu DNA polymerase (V100-R346) Taq DNA polymerase (E424-E832)
Taq DNA polymerase (1-291)Pfu DNA polymerase (H103-S334) Taq DNA polymerase (E424-E832); SEQ ID NO.:5
Taq DNA polymerase (1-291)Pfu DNA polymerase (V100-F389) Taq DNA polymerase (E424-E832)
Taq DNA polymerase (1-291)Pfu DNA polymerase (V100-F389) Taq DNA polymerase (V449-E832); SEQ ID NO.:6
Taq DNA polymerase (1-291)Pfu DNA polymerase (M1-F389) Taq DNA polymerase (V449-E832)
Of the above-mentioned polymerase chimeras the following were examined in more detail:
Taq DNA polymerase (M1-P291)E.coli DNA polymerase (Y327-H519) Taq DNA polymerase (E424-E832): point mutation A643G; Ile455Val (Taq Ec1) SEQ ID NO.:1
Taq DNA polymerase (M1-P291)E. coli DNA polymerase (Y327-G544) Taq DNA polymerase (V449-E832), (Taq Ec2) SEQ ID NO.:2
Taq DNA polymerase (M1-P291)Tne DNA polymerase (P295-E485) Taq DNA polymerase (E424-E832); silent mutation A1449C (Taq Tne1) SEQ ID NO.:3
Taq DNA polymerase (M1-P291)Tne DNA polymerase (P295-G510) Taq DNA polymerase (V449-E832); silent mutation C1767T (Taq Tne2) SEQ ID NO.:4
Taq DNA polymerase (1-291)Pfu DNA polymerase (V100-R346) Taq DNA polymerase (E424-E832), (Taq Pfu1) SEQ ID NO.:5
Taq DNA polymerase (1-291)Pfu DNA polymerase (V100-F389) Taq DNA polymerase (V449-E832), (Taq Pfu2) SEQ ID NO.:6
In order to select suitable DNA polymerases, multiple amino acid sequence alignments of available sequences of DNA polymerases and DNA binding proteins are established for example with the program GCG (Devereux et al., 1984, Nucl. Acids Res. 12, 387-395). In order to find a good alignment it is necessary to take into consideration the secondary structure predictions, known structure-based sequence alignments, known motifs and functionally essential amino acids as well as phylogenetic aspects. If the proteins are composed of functionally and structurally independent domains it is appropriate to firstly establish the amino acid sequence alignments with respect to the individual domains and only afterwards to combine them into a complete sequence alignment.
If homologous sequences are found whose tertiary structure is known, then it is possible to derive a 3D model structure from the homologous protein. The program BRAGI (Reichelt and Schomburg, 1988, J. Mol. Graph. 6, 161-165) can be used to make the model. The program AMBER (Weiner et al., 1984, J. Am. Chem. Soc. 106, 765-784) can be used for energy minimization of the structures of individual molecule regions and whole molecules and the program Procheck can be used to check the quality of the model. If only the Cxcex1 coordinates of the structure of the initial protein are available, the structure can for example be reconstructed using the program O (Jones et al., 1991, Acta Cryst. A47, 110-119). It is also possible to obtain Cxcex1 coordinates that are not available in the protein data bank but have been already published as a stereo picture by scanning the stereo picture and picking out the coordinates (for example using the program Magick) and calculating the z-coordinates (for example using the program stereo). Variants can be designed based on amino acid sequence alignments, based on 3D models or based on experimentally determined 3D structures.
In addition chimera variants were produced in which the domain with polymerase activity has reverse transcriptase activity. Examples of suitable polymerases are e.g. the polymerase from Anaerocellum thermophilum Ath or Thermus thermophilum Tth. The 3xe2x80x2-5xe2x80x2 exonuclease activity is inserted by a domain which is derived from another polymerase e.g. the Tne polymerase or the Pfu or Pwo polymerase. This chimera can additionally have 5xe2x80x2-3xe2x80x2 exonuclease activity in which case the domain with 5xe2x80x2 exonuclease activity can be derived from the first as well as from the second polymerase.
The recombinant hybrid polymerases HYB and HYBd5, like the DNA polymerase from Anaerocellum thermophilum, have a relatively strong reverse transcriptase activity in the presence of magnesium ions as well as in the presence of manganese ions. As shown in FIG. 22 the ratio of polymerase activity to reverse transcriptase activity is more favourable than with the Tth polymerase which is the most common and well-known enzyme of this type. This finding applies to the magnesium-dependent as well as to the manganese-dependent reverse transcriptase activity. It can be concluded from this that the polymerase domain which is derived from the Anaerocellum polymerase also exhibits full activity in the hybrid enzyme. The variant HYBd5 additionally has 3xe2x80x2-5xe2x80x2 exonuclease activity as shown in FIG. 21. This is inhibited by the presence of deoxynucleoside triphosphates as expected for the typical xe2x80x9cproof-reading activityxe2x80x9d. The exonuclease domain which is derived from the DNA polymerase from Thermotoge neapolitana is thus also active in the hybrid molecule. The ability to inhibit the exonuclease activity also demonstrates that both domains of the hybrid polymerase molecule interact and thus the hybrid polymerase is functionally very similar to the natural enzyme.
The production of domain exchange variants by genetic engineering can be achieved by PCR mutagenesis according to the SOE method (Horton et al. (1989) Gene 77, 61-68) or by the modified method (cf. scheme in the examples) with the aid of chemically synthesized oligodeoxynucleotides. The respective DNA fragments are separated on an agarose gel, isolated and ligated into the starting vector. pUC derivatives with suitable promoters such as pTE, pTaq, pPL, Bluescript can be used as starting vectors for E. coli. The plasmid DNA is transformed into an E. coli strain, for example XL1-blue, some clones are picked out and their plasmid DNA is isolated. It is also possible to use other strains such as Nova Blue, BL21 (DE), MC1000 etc. Of course it is also possible to clone into other organisms such as into yeast, plant and mammalian cells. A preselection of clones whose plasmid DNA is sequenced in the modified region is made by restriction analysis.
The gene expression in the target proteins can be induced by IPTG in many plasmids such as Pbtaq. When producing many different variants it is appropriate to establish a universal purification procedure. Affinity chromatography on Ni-NTA (nickel-nitrilotriacetic acid) agarose is well suited for this which can be used after attaching a His tag to the protein, for example by PCR. The protein concentrations can be determined with the protein assay ESL (Boehringer Mannheim) and contaminating side activities of the preparations can be determined as described for the commercially available Taq polymerase (Boehringer Mannheim). Polymerase, exonuclease activity and thermostability tests are carried out to further characterize the variants and the respective temperature optimum is determined. The polymerase activities of the chimeras can be determined in non-radioactive test systems for example by determining the incorporation rate of Dig-dUTP into DNase activated calf thymus DNA, or in radioactive test systems by for example determining the incorporation rate of xcex1-[32P]dCTP into M13 mp9 ssDNA. In order to determine the temperature optima of the polymerase activity of the chimeras, the polymerase reaction is carried out at different temperatures and the specific activities are calculated. The residual activities (i.e. the percentage of the initial activity without heat treatment) after heat treatment are measured in order to determine the thermal stabilities. The 3xe2x80x2-5xe2x80x2 exonuclease activity can be demonstrated by incorporation of a 5xe2x80x2-Dig-labelled primer which anneals to a DNA template strand starting at its 3xe2x80x2 end. The correction of 3xe2x80x2 mismatched primers and their extension (proof reading) can be shown by the extension of mismatched 5xe2x80x2-Dig-labelled primers which anneal to a template strand in the recognition sequence of a restriction enzyme (e.g. EcoRI). A cleavage with the restriction enzyme is only possible when the mismatch is corrected by the enzyme. The processivity can be examined by using variants in the PCR. If the enzyme is not sufficiently thermostable for use in PCR, a PCR can be carried out at the temperature optimum as the extension temperature with successive addition of enzyme. The exonuclease activity of the chimeras can be determined in a radioactive test system. For this a certain amount of the chimeric polymerases (usually 2.5 U) is incubated for 4 hours at various temperatures with labelled DNA (5 xcexcg [3H] DNA in the respective test buffers). dNTPs were optionally added at various concentrations (0-0.2 mM). After terminating the reaction the release of radioactively labelled nucleotides is determined.
A further subject matter of the present invention is the DNA sequence of the polymerase chimeras described above. In particular the DNA sequences SEQ ID NO.: 1-6 are a subject matter of the present invention. The present invention additionally concerns the amino acid sequences of the polymerase chimera described above. In particular the amino acid sequences SEQ ID NO.: 7-12 are a subject matter of the present invention. Moreover the DNA sequence SEQ ID NO.:17 is a subject matter of the invention.
Vectors which contain the above-mentioned DNA sequences are a further subject matter of the present invention. pBTaq (plasmid Pbtaq4_oligo 67 (Villbrandt (1995), dissertation, TU Braunschweig)) is a preferred vector.
The E. coli strains, in particular the strain Escherichia coli XL1-blue which contain the vector which carries the polymerase chimera gene are a further subject matter of the invention. The following strains were deposited at the DSM, xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbHxe2x80x9d, Mascheroder Weg 1b, D-38124 Braunschweig:
E.coli XL1 Bluexc3x97pBTaqEc1: TaqEc1 (SEQ ID NO:1) DSM No. 12053
E.coli XL1 Bluexc3x97pBTaqTne1:TaqTne1 (SEQ ID NO: 3) DSM No. 12050
E.coli XL1 Bluexc3x97pBTaqTne2:TaqTne2 (SEQ ID NO: 4) DSM No. 12051
E.coli XL1 Bluexc3x97pBTaqPfu1:TaqPfu1 (SEQ ID NO: 5) DSM No. 12052
The polymerase chimeras according to the invention are particularly suitable for amplifying DNA fragments e.g. for the polymerase chain reaction. A further application is for example to sequence DNA fragments.
A preferred vector for the Ath-Tne chimera is the following:
E.coli BL 21 (DE3) plysSxc3x97pETHYBR: HYBR
E.coli BL 21 (DE3) plysSxc3x97PETHYBR d5: HYBR d5
The E. coli strains which contain the vector which carries the polymerase chimera gene are a further subject matter of the invention. The following strains were deposited at the DSM, xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbHxe2x80x9d, Mascheroder Weg 1b, D-38129 Braunschweig: HYBR (DSM No. 12720); HYBR d5 (DSM No. 12719).
The production of the above-mentioned Ath-Tne chimeras is described for example in examples 8-11. The chimeras according to the invention which have RT activity are particularly suitable for the reverse transcription of RNA.
A further subject matter of the present invention is a kit for amplifying DNA fragments which contains at least one of the polymerase chimeras according to the invention.